1. Field of the Invention
The present invention relates to oligonucleotides that can be used for detection and identification of mycobacteria. More particularly, the present invention identifies the nucleotide sequence of ITS (Internal Transcribed Spacer region) of non-tuberculosis mycobacteria, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium porcinum, Mycobacterium acapulcensis and Mycobacterium diernhoferi, and using the nucleotide sequences, it provides oligonucleotide primers or probes used for detection and identification of mycobacteria.
2. Description of the Related Art
A Even though the number of patients of tuberculosis has steadily decreased in these days, about 8 million patients have come out and about 3 million patients died of tuberculosis in a year. Moreover, in underdeveloped countries, inadequate treatment and lack of drugs for tuberculosis increase chronic carriers of drug-resistant bacteria. In 1980""s, spread of AIDS has increased patients of tuberculosis even in advanced countries. In this condition, it is expected that about twelve million patents of tuberculosis would newly come out in the year 2000 (J. P. Natain, M. C. Raviglione, and A. Kochi, Tubercle and Lung Disease, 73: 311-321, 1992; Murray C J L. And Lopez A D. The global burden of disease. Global burden of disease and injury series. Vol. 1. Cambridge, Mass.: Harvard University Press, 1996, p349-350; Global Tuberculosis Programme: Anti-tuberculosis drug resistance, WHO Report 1997: World Health Organization. 1997).
In 1950""s, it was reported that non-tuberculosis mycobacteria (NTM) has been able to cause diseases in human. After the report that M. avium complex (MAC) would bring about systemic disease in the patients of AIDS in 1980, non-tuberculosis mycobacteria have taken an interest. Diseases caused by non-tuberculosis mycobacteria are similar to tuberculosis in clinical condition and general pathological view. Non-tuberculosis mycobacteria are distributed in a wide range of living environment, and it is difficult to judge whether they have pathogenicity or not in clinical test sample. Further, since they have resistance to a number of drugs for tuberculosis, the infection is hard to treat and the recurrence rate is high. The infection of non-tuberculosis mycobacteria should be treated by other means than for tuberculosis, and therefore, accurate and fast method of detecting and identifying non-tuberculosis mycobacteria is required. The accurate and fast method of detecting and identifying both TB complex and NTM is also needed for effective treatment and management of tuberculosis.
Many a method has been developed to diagnose mycobacterial infection and to detect and identify mycobacteria strains. Among them, the following methods are used at present;
The first is a microbiological method, that is, smearing, staining and culturing test. However, this method is not suitable for mycobacteria, since they have long generation term and need long culturing time. Further, such pathogenic microorganism as mycobacteria is dangerous to infect the personnel in culture room;
The second is a PCR (Polymerase Chain Reaction) method. It is highly sensitive and specific to the mycobacteria and very useful to detect mycobacteria which have a long culturing time. Especially, it does not require a culturing process but uses a small amount of DNA to be amplified, therefore, only a small amount of pathogens in test sample is enough to detect and identify mycobacteria. Many a PCR process has been introduced with different target DNAs each other, and IS6110 and 16S rRNA are often used as the target (Bauer J, Andersen A B, Kremer K, and Miorner H, Usefulness of spoligotyping to discriminate IS6110 low-copy-number Mycobacterium tuberculosis complex strains cultured in Denmark, 1999, J. Clin. Microbiol. 37: 2602-2606; Troesch, A., H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M. Kaplan, P. Cros and C. Mabilat. 1999, Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays, J. Clin. Microbiol. 37: 49-55);
The third is a physico-chemical process, in which lipid component in mycobacteria has been detected by HPLC, GC or mass spectrophotometer. This method is very specific but rquires expensive equipments;
The fourth is a method of detecting mycobacteria composition by serological method. This method uses a coagulation reaction of latex particles or blood corpuscles adsorbed with antibody to mycobacterial antigen or enzyme-linked immunological method in which enzyme is linked with antibody. It is, however, very sensitive only to be proceeded within a limited place. Further, it is difficult for this method to distinguish present infection from previous infection;
The next method to detect mycobacteria consists of infecting mycobacteria with mycobacteriophage L5 inserted with luciferase gene, and inspecting luminescence by luciferin in medium (W. R. Jacobs, R. G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G. J. Sarkis, G. F. Hatful, and B. R. Bloom. 1993, Science 260: 819-822); and
The last is a method of detecting and identifying mycobacteria by hybridization of oligonucleotide (A. Troesch, H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M. Kaplan, P. Cros and C. Mabilat. 1999. J. Clin. Microbiol 37: 49-55).
Besides Mycobacierium avium complex (MAC) described above, M. Fortuitum, M. chelonae complex, M. terrae and M. vaccae are also known as non-tuberculosis mycobacteria. Among them, M. chelonae complex are classified into M. chelonae and M. abscessus, and there is no means to distinguish one from the other at present.
To solve the problems in the prior method of detection and identification of mycobacteria, it is an objective of the present invention to provide specific oligonucleotides as probes or primers for PCR which can be used to detect mycobacteria, to distinguish TB complex from NTM, and to identify species of mycobacteria with an accuracy and effectiveness.
To accomplish the above objective, the present invention provides a DNA of ITS (Internal Transcribed Spacer region) of Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium porcinum, Mycobacterium acapulcensis and Mycobacterium diernhoferi genes set forth in SEQ ID NOs: 1 to 9.
Further, the present invention provides, as a primer for PCR or a probe for hybridization, an oligonucleotide for detection of mycobacteria set in forth in one of SEQ ID NOs: 10 to 14;
an oligonucleotide for distinction of TB complex from NTB among mycobacteria set in forth in one of SEQ ID NOs: 15 to 23;
an oligonucleotide for detection and identification of MAC (Mycobacterium avium and Mycobacterium intracellulare) set in forth in one of SEQ ID NOs: 24 to 27;
an oligonucleotide for detection and identification of Mycobacterium fortuitum set in forth in one of SEQ ID NOs: 28 to 38;
an oligonucleotide for detection and identification of Mycobacterium chelonae set in forth in one of SEQ ID NOs: 39 to 46;
an oligonucleotide for detection and identification of Mycobacterium abscessus set in forth in one of SEQ ID NOs: 47 to 52;
an oligonucleotide for detection and identification of Mycobacterium vaccae set in forth in one of SEQ ID NOs: 53 to 64;
an oligonucleotide for detection and identification of Mycobacterium flavescens set in forth in one of SEQ ID NOs: 65 to 72;
an oligonucleotide for detection and identification of Mycobacterium gordonae set in forth in one of SEQ ID NOs: 73 to 77;
an oligonucleotide for detection and identification of Mycobacterium terrae set in forth in one of SEQ ID NOs: 78 to 100;
an oligonucleotide for detection and identification of Mycobacterium scrofulaceum set in forth in one of SEQ ID NOs: 101 to 108;
an oligonucleotide for detection and identification of Mycobacterium kansasii set in forth in one of SEQ ID NOs: 109 to 112;
an oligonucleotide for detection and identification of Mycobacterium szulgai set in forth in one of SEQ ID NOs: 113 to 116;
an oligonucleotide for detection and identification of Mycobacterium marinum and Mycobacterium ulcerans set in forth in one of SEQ ID NOs: 117 to 119;
an oligonucleotide for detection and identification of Mycobacterium gastri set in forth in one of SEQ ID NOs: 120 to 123;
an oligonucleotide for detection and identification of Mycobacterium xenopi set in forth in one of SEQ ID NOs: 124 to 133;
an oligonucleotide for detection and identification of Mycobacterium genavense set in forth in one of SEQ ID NOs: 134 to 141;
an oligonucleotide for detection and identification of Mycobacterium malmoense set in forth in one of SEQ ID NOs: 142 to 146;
an oligonucleotide for detection and identification of Mycobacterium simiae set in forth in one of SEQ ID NOs: 147 to 153;
an oligonucleotide for detection and identification of Mycobacterium smegmatis set in forth in one of SEQ ID NOs: 154 to 165;
an oligonucleotide for detection and identification of Mycobacterium shimoidei set in forth in one of SEQ ID NOs: 166 to 172;
an oligonucleotide for detection and identification of Mycobacterium habana set in forth in one of SEQ ID NOs: 173 to 180;
an oligonucleotide for detection and identification of Mycobacterium farcinogen set in forth in one of SEQ ID NOs: 181 to 189;
an oligonucleotide for detection and identification of Mycobacterium asiaticum set in forth in one of SEQ ID NOs: 190 to 193;
an oligonucleotide for detection and identification of Mycobacterium porcinum set in forth in one of SEQ ID NOs: 194 to 205;
an oligonucleotide for detection and identification of Mycobacterium acapulcensis set in forth in one of SEQ ID NOs: 206 to 215;
an oligonucleotide for detection and identification of Mycobacterium diernhoferi set in forth in one of SEQ ID NOs: 216 to 227;
an oligonucleotide for detection and identification of Mycobacterium paratuberculosis set in forth in one of SEQ ID NOs: 228 to 240; and
an oligonucleotide for detection of Mycobacteria sp. set in forth in SEQ ID NO: 241.
In the prior method of detecting and identifying mycobacteria using PCR, only one or two strains can be detected. According to the present invention, however, almost all of mycobacteria strains can be detected and identified, since primers and probes of the present invention have been designed form DNA sequences of ITS of mycobacteria. ITS has more polymorphic region than 16S rRNA has and ITS also has conserved region, therefore, it is highly effective as a target DNA for distinction of genotype (Gurtler, V., and V. A. Stanisich, 1996, New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiol. 142: 3-16).
The inventors identified DNA sequences of ITS of non-tuberculosis mycobacteria whose DNA had not yet been sequenced, such as Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium porcinum, Mycobacterium acapulcensis and Mycobacterium diernhoferi. Using the DNA sequences, oligonucleotides for primers or probes have been designed for detecting and identifying the mycobacteria. Further, referring to the information on DNA sequence of other mycobacteria disclosed in GenBank, and analyzing the information with multi-alignment and blast, distinctive regions of polymorphism were selected to design oligonucleotides for primers or probes to detect and identify mycobacteria. The oligonucleotides have been confirmed to detect and identify mycobacteria by specific hybridization and amplification with species-specific and genus-specific primers of PCR.
That is, the oligonucleotide probes of the present invention, attached to solid substrate, are hybridized only with nucleotide sequence in ITS of specific mycobacteria, and therefore, they can detect and identify the specific mycobacteria sensitively. Further, the oligonucleotide primers of identical nucleotide sequence with the above probes can also detect and identify the specific mycobacteria by amplification in PCR. Using the oligonucleotide primers or probes made from ITS of mycobacteria, it is possible to detect mycobacteria, distinguish TB complex from NTM, and identify mycobacteria species accurately and effectively.